Understanding the mechanisms of regulation of gene expression is essential to a comprehension of development and differentiation, and thus, the disorders that arise when these processes go awry. Steroid hormone regulated gene expression provides an excellent model system with which to investigate regulatory mechanisms. I have chosen to take advantage of a partially characterized system regulated by glucocorticoid hormones, that of the mouse mammary tumor virus (MMTV), to investigate the fine structure details of the molecular interaction of glucocorticoid receptor with the regulatory elements of the promoter region. (i) I will utilize a new and novel approach to oligonucleotide-directed mutagenesis to generate a library of mutant DNA regulatory elements. (ii) These elements will be examined for biological activity using a rapid, sensitive DNA-mediated gene transfer assay and a vector I have constructed expressly for this purpose. (iii) The cloned elements will be assayed for their ability to be specifically recognized by glucocorticoid receptor. Further fine structure mapping will be performed employing methylation protection experiments to identify specific points of receptor-DNA interaction in the wild type and mutant sequences. (iv) The results of (ii) and (iii) will be used to assemble maps of the residues critical for receptor binding and for hormone response. These data will address the question of whether specific receptor binding is necessary and sufficient for biological activity of the hormone response element. (v) I will determine if any of the mutant elements exhibit a differential ability to bind glucocorticoid receptor vis a vis the progesterone receptor. Progesterone receptor has recently been reported to bind in vitro to the same region of the hormone response element as the glucocorticoid receptor even though progesterone is biologically inactive as a direct inducer of MMTV transcription. (vi) I will determine the minimal sequence that comprises a functional element. These same experiments will also test the hypothesis that the activity of the hormone response element is influenced by other nearby transcription control elements.